1995 年 117 巻 4 号 p. 836-844
In the previous study involving a yeast expression system, a high molecular mass extracellular domain of human tissue factor (denoted as sTFα) with a high content of mannose residues was produced in abundance and 37 kDa sTFβ was obtained in a low yield [Shigematsu et al. (1992) J. Biol. Chem. 267, 21329-21337]. To obtain sTFβ in a high yield, we constructed four kinds of mutant sTF with partial or total replacement of the N-potential glycosylation Asn residues with Ala, and expressed them in yeast. We found that the yield of the β form of the Asn137-to-Ala mutant (designated as sTFβNNA) was threefold higher (3mg/liter) than that of the wild type, suggesting that the replacement of one of the three potential N-glycosylation Asn residues with Ala could be a good way to minimize the addition of mannose repeats. Since it has been reported that calcium ions are required for the effective hydrolysis of peptidyl substrates by the factor VIIa-sTF complex, it is believed to be essential for the expression of full protease activity. Here, we report the enzymatic characterization of a factor VIIa-sTFβNNA complex cross-linked with a homobifunctional reagent, bis(sulfosuccinimidyl) suberate. The factor VIIa-sTFβNNA complex cross-linked in the presence of 5mM calcium ions or 50mM EDTA was purified. The cross-linked complex did not show factor X activation in the presence of phospholipids. However, it showed essentially the same activity toward peptidyl substrates as before cross-linking, even in the presence of EDTA. The kinetic constants of the cross-linked complexes in the presence of 5mM calcium and 50mM EDTA were similar, indicating that once factor VIIa was cross-linked with sTF, calcium ions were required no more for its activity. Therefore, calcium ions are not required for full catalytic activity of the factor VIIa-sTF complex toward synthetic substrates once the bi-molecular complex has formed.
